[go: up one dir, main page]

WO2004073633A2 - Methodes et compositions permettant de moduler le developpement de cellules souches - Google Patents

Methodes et compositions permettant de moduler le developpement de cellules souches Download PDF

Info

Publication number
WO2004073633A2
WO2004073633A2 PCT/US2004/004639 US2004004639W WO2004073633A2 WO 2004073633 A2 WO2004073633 A2 WO 2004073633A2 US 2004004639 W US2004004639 W US 2004004639W WO 2004073633 A2 WO2004073633 A2 WO 2004073633A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
bmp
cells
signaling pathway
substance
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2004/004639
Other languages
English (en)
Other versions
WO2004073633A3 (fr
Inventor
Seung Kim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leland Stanford Junior University
Original Assignee
Leland Stanford Junior University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leland Stanford Junior University filed Critical Leland Stanford Junior University
Priority to US10/545,582 priority Critical patent/US20070155661A1/en
Publication of WO2004073633A2 publication Critical patent/WO2004073633A2/fr
Anticipated expiration legal-status Critical
Publication of WO2004073633A3 publication Critical patent/WO2004073633A3/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/19—Growth and differentiation factors [GDF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • Diabetes mellitus is a major cause of morbidity and mortality worldwide, and incidence rates of type I and type II DM are increasing.
  • type I DM destruction of insulin-producing pancreatic islets leads to a prolonged illness often culminating in devastating multisystem organ failure and early mortality.
  • Clinical trials demonstrate that tight glucose regulation can prevent the development of diabetic complications, but attempts to achieve this regulation by exogenous insulin administration are only partially successful.
  • Stem cells including embryonic stem (ES) cells and various adult stem cells provide a promising potential means for cell-replacement therapy in human diseases.
  • Stem cells may provide serve as an inexhaustible source for the production of replacement islets for transplantation in diabetic humans.
  • conditions to produce stably-differentiated functional insulin-producing cell compositions (ICCs) with stem cells generally, and particularly ES stem cells, have not been developed to a clinically satisfactory level.
  • Methods to provide a renewable source of replacement islets from stem cells could transform therapeutics in DM.
  • methods for stimulating the production of insulin producing cells in patients could also have significant therapeutic effects.
  • the application provides methods for manipulating the development of insulin producing cells and beta cell precursor cells by activating or antagonizing a BMP signaling pathway. Such methods may be applied, for example, to cultured cells or to subjects in need of improved pancreatic function.
  • the application provides methods for increasing the production of insulin in a cell composition by culturing the cell composition in the presence of a BMP family member, such as a GDF8/GDF11 subfamily member, or an activator of a BMP signaling pathway.
  • a BMP family member such as a GDF8/GDF11 subfamily member, or an activator of a BMP signaling pathway.
  • the BMP family member has one or more of the following characteristics: an amino acid sequence that is at least 80% identical to a human GDF11 or GDF8; ability to bind to an ActRIIA and/or ActRIIB receptor; an ability to increase Smad2 and/or Smad3 phosphorylation in a stem cell; an ability to increases expression of a gene that is positively regulated by Smad4.
  • the application provides methods for increasing insulin production in a cell composition comprising stem cells, the method comprising contacting the cell composition with a substance that stimulates an ActRIIA and/or ActRIIB signaling pathway.
  • Activators of an ActRIIA and/or ActRIIB signaling pathway may, for example, cause an increase in Smad2 and/or Smad3 function or phosphorylation, or increase expression of a gene that is positively regulated by Smad4.
  • a BMP signaling pathway may be activated by causing overexpression of one or more positive regulators, such as Smad2, Smad3, or a BMP family member.
  • a BMP signaling pathway may also be activated by inhibiting an inhibitor, such as noggin or chordin.
  • the cell composition comprises beta cell precursor cells.
  • the cell composition is derived from embryonic stem cells that have been cultured in the presence of a retinoid.
  • the application provides a method for promoting the maturation of beta cell precursor cells, the method comprising contacting the beta cell precursor cells with a BMP family member.
  • the application provides methods for increasing the number of beta cell precursor cells in a cell composition by culturing the cell composition in the presence of an antagonist of a BMP signaling pathway.
  • the antagonist of a BMP signaling pathway provides methods for increasing the number of beta cell precursor cells in a cell composition by culturing the cell composition in the presence of an antagonist of a BMP signaling pathway.
  • BMP signaling pathway may be a secreted polypeptide, such as noggin, chordin or follistatin that binds directly to a BMP family member.
  • the application provides cell culture methods that employ sequential inhibition and activation of a BMP signaling pathway to obtain insulin- producing cells.
  • the cells may be cultured in the presence of a noggin, chordin or follistatin and then cultured in the presence of a BMP family member, such as a member of the GDF8/GDF11 subfamily.
  • the stem cells are islet precursor cells.
  • the application provides methods for ameliorating a condition associated with insufficient pancreatic function by administering a BMP family member, such as a GDF8/GDF11 subfamily member, or an activator of a BMP signaling pathway.
  • a BMP family member such as a GDF8/GDF11 subfamily member
  • an activator of a BMP signaling pathway Suitable subjects include those that have been diagnosed with type I or type II diabetes.
  • the application provides methods for increasing the production of beta cell precursor cells in a subject, the method comprising administering to the subject a substance that inhibits a BMP. signaling pathway, such as a noggin, chordin or follistatin.
  • a BMP signaling pathway
  • sequential use of BMP pathway antagonists and activators may be desirable.
  • Insulin production in a subject may also be enhanced by administering a composition comprising insulin producing cells produced according to a method of the application.
  • the application provides methods for assessing the effectiveness of a test agent for modulating the development of insulin producing cells.
  • a method may comprise: forming a mixture comprising the test agent and a BMP pathway polypeptide; and detecting binding between the test agent and the polypeptide, wherein a test agent that binds the BMP pathway polypeptide specifically and w ith a n a ff ⁇ nity o f a 1 1 east 1 0 "4 M h as a n increased 1 ikelihood o f being effective for modulating the development of insulin producing cells.
  • a cell is cultured with the test agent; and an activity of a BMP signaling pathway is detected, wherein a test agent that increases the activity of a BMP signaling pathway has an increased likelihood of being effective for increasing insulin production in a cell.
  • Test agents for promoting formation of beta cell precursor cells may similarly be tested by determining whether they cause a decreases in the activity of a BMP signaling pathway.
  • Cells for use in such assays preferably express an ActRIIA and/or ActRILB receptor.
  • assay cells comprise a reporter gene, wherein expression of the reporter gene is positively or negatively regulated by a BMP signaling pathway.
  • the activity of a BMP signaling pathway may be assessed in a variety of ways, including measuring expression of a reporter gene regulated by a BMP signaling pathway or by measuring a change in one or more components of the BMP signaling pathway. For example, phosphorylation of one or more pathway proteins, such as ActRIIA, ActRIIB, Smad2 and Smad3, may be evaluated.
  • FIG. 1 Gdfl l expression in the pancreatic islet progenitor cell niche.
  • El 3.5 pancreatic cells expressing ngn3 ((blue staining) are detected by antisense RNA in situ hybridization.
  • ngn3 + cells are found adjacent and contiguous to Gdfll- expressing epithelial cells stained brown with an antiserum specific for Gdfl l.
  • FIG. 1 Defective pancreatic development in embryos deficient for Gdfl l. Gdfll + indicates that the observed phenotype is indistinguishable in Gdfll +/+ and Gdfll +/ ⁇ embryos.
  • (a,b) Whole mounted preparations of pancreas from Gdfll +/ ⁇ and Gdfir' ⁇ mice at postnatal day 1.
  • (c) Pancreatic mass unaffected in Gdfll ⁇ ' ⁇ mice. Data are shown as the average measurements from at least 4 mice of indicated genotypes ⁇ standard error of the mean.
  • Figure 3 D effete p ancreatic i slet ⁇ -cell and ⁇ -cell n umbers i n G dfl 1 d mice, (a-c) Immunohistochemical detection of insulin (brown staining) at E17.5 in mice with the indicated genotypes, (d) Quantification of El 7.5 ⁇ -cell mass by point- counting morphometry in Gdfl l +/+ , Gdfl l + ⁇ , and Gdfl l _/ ⁇ pancreata.
  • FIG. 5 Pancreatic defects in E17.5 Smad2 mutant embryos.
  • El 7.5 in wildtype pancreas only small clusters of ngn3 cells are detected.
  • ngn3 expression is abnormally persistent in clustered periductal epithelial cells.
  • e,f Nkx ⁇ .l expression (brown nuclei marked by arrows).
  • h ngn3 + nuclei (Cy3, red) adjacent to insulin "1" cells (FITC, green). These images are representative of 5 or more animals per genotype.
  • Conditions #1-9 exposure of human embryoid bodies to 2 micromolar retinoic acid (RA) for 7 days, followed by exposure for 7 days to (1) two micromolar RA, (2) lOmM Nicotinamide, (3) 10 micromolar LY294002, (4) Both lOmM Nicotinamide and 10 micromolar LY294002, (5) 10 ng/mL GDF8, (6) 10 nM Nicotinamide and 10 ng/mL GDF8, (7) lOmM Nicotinamide and 10 micromolar LY294002 and 10 ng/mL GDF8, (8) 2 nM activin A, or (9) lOmM Nicotinamide and 2 nM activin A.
  • RA retinoic acid
  • Conditions #10-18 exposure of human embryoid bodies to 100 nM retinoic acid (RA) for 7 days, followed by exposure for 7 days to (10) 100 nM RA, (11) lOmM Nicotinamide, (12) 10 micromolar LY294002, (13) Both lOmM Nicotinamide and 10 micromolar LY294002, (14) 10 ng/mL GDF8, (15) 10 nM Nicotinamide and 10 ng/mL GDF8, (16) lOmM Nicotinamide and 10 micromolar LY294002 and 10 ng/mL GDF8, (17) 2 nM activin A, (18) lOmM Nicotinamide and 2 nM activin A. Results are average of triplicate samples.
  • an “ActRIIA or ActRIIB signaling pathway” refers to polypeptides and polypeptide interactions that participate in transducing or otherwise effectuating changes in the properties of a cell upon stimulation of an ActRIIA and/or ActRIIB receptor (e.g. by contacting the receptor with a natural ligand such as a GDF8 or GDF11), including the receptors themselves.
  • An example of an ActRIIA or ActRIIB signaling pathway is the ActRII-ActRI-Smad2/3-Smad4 transcriptional regulation pathway.
  • adult stem cell is used herein to refer to a stem cell obtained from any non-embryonic tissue.
  • stem cells may be sorted into two categories: “embryonic” and “adult” (or, equivalently, “non-embryonic”).
  • Beta cell precursor cells are cells having generally mesenchymal, non-cell- cell adherent qualities that form insulin producing cells under appropriate conditions.
  • a "cell composition” is any composition of matter generated by human manipulation that comprises viable cells as a substantial component.
  • a n "enriched c ell composition” is a cell composition comprising a substantially greater purity (i.e. at least twice as pure) of a recognizable cell type than is found in any natural tissue.
  • a “pure cell composition” is a cell composition that comprises at least about 75%, and optionally at least about 85%, 90% or 95% of a recognizable cell type.
  • a recognizable cell type is generally one that has a reasonably uniform morphology, a characteristic set of two or more molecular markers and a functional characteristic.
  • a cell composition may comprise, in addition to cells, essentially any component(s) that are compatible with the intended use for the cell composition.
  • a cell composition may include media, growth factors, pharmaceutically acceptable excipients, preservatives, a solid or semi-solid substrate, a porous matrix or scaffold, nonviable cells or a therapeutic agent.
  • culturing includes exposing cells to any condition. While “culturing” cells is often intended to promote growth of one or more cells, “culturing” cells need not promote or result in any cell growth, and the condition may even be lethal to a substantial portion of the cells.
  • a later cell is "derived" from an earlier cell if the later cell is descended from the earlier cell through one or more cell divisions. Where a cell culture is initiated with one or more initial cells, it may be inferred that cells growing up in the culture, even after one or more changes in culture condition, are derived from the initial cells. A later cell may still be considered derived from an earlier cell even if there has been an intervening genetic manipulation.
  • a member of the "GDF8/GDF11 subfamily” is a polypeptide (or an encoding nucleic acid) comprising an amino acid sequence that is at least 80% identical to an amino acid sequence of a mature, naturally occurring GDF8 or GDF11 polypeptide, such as the mature human or mouse GDF8 and GDF11 sequences, a fragment thereof. A member of the GDF8/GDF11 subfamily should also retain the ability to stimulate a receptor-mediated signaling pathway.
  • islet precursor cell refers to any cell that has differentiated so as to be recognizably of pancreatic lineage and that differentiates under appropriate conditions to give rise to beta cell precursor cells.
  • percent identical refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Percent identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. Expression as a percentage of identity refers to a function of the number of identical amino acids or nucleic acids at positions shared by the compared sequences.
  • Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ.
  • FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings.
  • ENTREZ is available through the
  • the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
  • an alignment program that permits gaps in the sequence is utilized to align the sequences.
  • the Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173- 187 (1997).
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences.
  • An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer.
  • MPSRCH uses a Smith- Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors.
  • Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
  • stem cell refers to an undifferentiated cell which is capable of proliferation and giving rise to at least one more differentiated cell type.
  • Totipotent stem cells are stem cells that are capable of giving rise to any cell type of the organism from which the stem cells were obtained.
  • Pluripotent stem cells are stem cells that are capable of giving rise to cells of the three major embryonic lineages, the endoderm, mesoderm and ectoderm.
  • Multipotent stem cells are stem cells that are capable of giving rise to more than one type of more differentiated cell.
  • stem cell is also intended to include cells of varying developmental potential that may be obtained by somatic cell nuclear transfer or by causing a differentiated cell to undergo de-differentiation.
  • a stem cell is named by the tissue from which it was obtained.
  • a "neural stem cell” is a stem cell obtained from a neural tissue (or a fluid, such as cerebrospinal fluid that is in contact with neural tissue)
  • a “neuroendocrine stem cell” is a stem cell derived from a neuroendocrine tissue, such as the adrenal gland or the pituitary gland, but specifically excluding the pancreas.
  • An “embryonic stem cell” is a stem cell obtained from an embryo. Many "tissues" are complex and actually contain several different stem cell types.
  • the skin may be considered a tissue, but skin contains neural stem cells of the peripheral nervous system, skin stem cells from the dermis, and stem cells from the blood circulating through the skin. Accordingly, in determining the classification of a stem cell, the true origin, including sub-tissue structures, should be carefully considered.
  • a "stem cell line” is an enriched or pure cell composition comprising a recognizably distinct stem cell type that, when cultured in appropriate c onditions, self-propagates.
  • the application provides methods for increasing insulin- production i n a s Ampomy cell c omposition by e ulturing t he c ell c omposition i n t he presence of a BMP family member or other activator of a BMP signaling pathway.
  • Suitable cell populations will generally include any cell populations containing stem cells that retain the ability to develop into insulin-producing cells.
  • stem cell includes cells that are islet cell or beta cell precursors.
  • a suitable cell population comprises a cell that has undergone an epithelial to mesenchymal cell type shift, but has not undergone a reorganization back to the epithelial cell traits that are characteristic of mature pancreatic beta cells.
  • pancreatic islets involves a shifting of cell characteristics from epithelial to mesenchymal and then back to epithelial again.
  • the cells of the pancreatic duct have epithelial characteristics, meaning that the cells adhere closely, possibly through tight junctions, to form sheets.
  • cells of the duct become mesenchymal in character, meaning that the adhesions to neighboring cells dissolve and the cells move away from the two dimensional sheet. Then the cells develop adhesive, epithelial qualities again and form into islets, separated from the original ductal cell sheet.
  • the final steps of islet cell maturation are marked by a cessation of proliferation.
  • Members of the BMP family may encourage the formation of epithelial structures, thereby directing the dispersed mesenchymal cells (e.g. beta cell precursors) to form multicellular islet structures.
  • Members of the BMP family also inhibit proliferation, in part by causing expression of inhibitors of cyclin-dependent kinases (cdks), such as pl5, pl7, pl8, p21, p27, or p57.
  • cdks cyclin-dependent kinases
  • Inl ibitors of the BMP signaling pathway may stimulate the formation of beta cell precursor cells in two ways. First, such inhibitors may stimulate epithelial cells of the duct to adopt mesenchymal characteristics, and second, such inhibitors may prevent formation of the islet epithelial structures.
  • a "BMP family member” may be selected from the naturally occurring members of the TGF-beta/BMP family, and may also be a functional analog thereof. Naturally occurring members of this family are characterized by common sequence homology and as being proteins that are translated as preproprotein precursors.
  • a preprotein precursor contains an N-terminal signal peptide, a prodomains and a mature portion. The mature portion contains six to nine conserved cysteine residues that participate in forming intermolecular disulfide bonds, although a few members (e.g. GDF-9, BMP- 15, GDF-3, lefty-1 and lefty-2) contain a serine substitution for a cysteine involved in disulfide bond formation.
  • GDF-9, BMP- 15, GDF-3, lefty-1 and lefty-2 contain a serine substitution for a cysteine involved in disulfide bond formation.
  • BMP family members including the following: TGF-beta2, TGF-beta3, TF-betal, GDF-15, GDF-9, BMP-15, BMP-16, BMP-3, GDF-10, BMP-9, BMP-10, GDF-6, GDF-5, GDF-7, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, BMP-4, GDF-3, GDF-1, GDF11, GDF8, Activins betaC, betaE, betaA and betaB, BMP-14, GDF-14, MIS, Inhibin alpha, Lefty 1, Lefty2, GDNF, Neurteurin, Persephin and Artemin.
  • BMP family member is intended to include variant forms of any of the naturally occurring polypeptides, including fusion proteins, truncated proteins, and mutant forms, although only if such variants retain the ability to stimulate a receptor- mediated signaling pathway and further have sufficient sequence similarity to a naturally occurring family member as to be recognizable using a sequence comparison algorithm such as BLAST or PILEUP.
  • BMP family member is also intended to refer both to single polypeptides and homo- and hetero- dimers or other multimeric forms, as w ell as nucleic acids encoding polypeptides that are BMP family members.
  • GDF8/GDF11 subfamily e.g.
  • GDF8 GDF11
  • the "Activin C subfamily” e.g. Activins betaC, betaE, and BMP-14
  • the "Activin A subfamily” e.g. Activin betaA, b etaB
  • the "TGF-beta subfamily” e.g. T GF-beta2, T GF-beta3, TF-betal
  • the "BMP-5 subfamily” e.g. BMP-5, BMP-6, BMP-7
  • the "BMP-8 subfamily” e.g. BMP-8
  • the "GDF-6 subfamily” e.g. GDF-5, GDF-6, GDF-7
  • the "BMP-9 subfamily” e.g.
  • BMP-9, BMP-10 the "BMP-3 subfamily” (e.g. BMP-3, GDF-10), the "GDF-9 subfamily” (e.g. GDF-9, BMP-15) and the "GDF-15 subfamily” (e.g. GDF-15).
  • a BMP family member may be replaced by, or used in conjunction with, a BMP signaling pathway activator.
  • a BMP signaling pathway activator is a compound that stimulates one or more of the polypeptides or interactions that participate i n t ransducing o r o therwise e ffectuating c hanges i n t he p roperties o f a cell in response to a BMP family member.
  • a BMP signaling pathway includes BMP family members themselves.
  • An example of a BMP signaling pathway is the GDFl l-ActRII-ActRI-Smad2/3-Smad4 transcriptional regulation pathway.
  • the BMP family member binds to the extracellular ligand binding domain portion of the ActRII receptor and then forms a complex with ActRI, leading to the inhibition of the Smad7 negative regulator and phosphorylation of the Smad2/Smad3 complex.
  • the Smad2/Smad3 complex associates with Smad4 to regulate expression of certain genes.
  • activation of a BMP signaling pathway leads to activation of small inhibitors of cyclin-dependent kinases (cdks), such as pi 5, pi 7, pi 8, p21, p27 and p57.
  • a BMP signaling pathway activator is also a compound that antagonizes an inhibitor of a BMP signaling pathway, such as an anti-noggin or anti- chordin antibody.
  • certain method embodiments of the application comprise culturing s tem c ells, p referably e mbryonic s tern cells, to a d esired s tage and t hen contacting the cells with a BMP family member, preferably a GDF8/GDF11 subfamily member.
  • a BMP family member preferably a GDF8/GDF11 subfamily member.
  • Public database references for preferred GDF8/GDF11 are set forth in Table 1, and such references provide guidance as to the mature and pro- polypeptide sequences.
  • the database entries listed in Table 1 are incorporated herein by reference, in their entirety.
  • human embryonic stem cells are cultured in the presence of a retinoid compound, such as all-trans retinoic acid and then cultured in the presence of a GDF8/GDF11 subfamily member, thereby generating a cell composition comprising insulin-producing cells.
  • a retinoid compound such as all-trans retinoic acid
  • the application provides methods for increasing the number of beta cell precursor cells in a cell composition by culturing cells in the presence of an inhibitor of a BMP pathway.
  • Inhibitors of a BMP pathway include noggins, chordins, follistatins, twisted gastrulation (TSG), Dan, Cerberus and Xenopus nodal related 3 (Xnr3), to name only a few.
  • TSG twisted gastrulation
  • Dan Dan
  • Cerberus Cerberus
  • Xenopus nodal related 3 Xenopus nodal related 3
  • antibodies that bind to BMP family members and prevent receptor activation may also be used as BMP p athway i nhibitors.
  • P ublic d atabase r eferences for p referred B MP p athway inhibitors are set forth in Table 2, and such references provide guidance as to the mature and pro-polypeptide sequences.
  • the database entries listed in Table 2 are incorporated herein by reference, in their entirety.
  • a cell composition comprising stem cells may be cultured sequentially with an inhibitor of BMP signaling and then with an activator of BMP signaling, thereby obtaining insulin-producing cells.
  • Stem cells for use in the methods disclosed herein may b e essentially any stem cell that has not lost the potential to become a pancreatic hormone-producing cell.
  • the term "stem cell” as used herein refers to an undifferentiated cell which is capable of proliferation and giving rise to at least one more differentiated cell type.
  • Stem cells may be totipotent, pluripotent stem cells or multipotent. Stem cells may also be obtained by somatic cell nuclear transfer or by causing a differentiated cell to undergo de-differentiation.
  • stem cells for use with the disclosed methods may be impure, such as stem cells nested in a tissue or in a suspension obtained from a tissue sample. It is now widely believed that most adult tissues include small populations of stem cells, as that term is used herein. Stem cells may also be enriched from tissue samples, and may optionally be purified stem cells. Stem cells may also be used from stem cell lines, and preferably from well- characterized and established stem cell lines. Tissue may be embryonic or "adult” as the term is used herein, including fetal, infant, child and mature animal tissue. Cells need not be obtained from a tissue, and other cell-containing sources that are not g enerally considered "tissues" m ay be employed ( e.g. c erebrospinal fluid and mucus or secreted fluids of the lung or gut).
  • a stem cell for use in a disclosed method is an embryonic stem cell.
  • mouse embryonic stem cells include: the JM1 ES cell line described in M. Qiu et al., Genes Dev 9, 2523 (1995), and the ROSA line described in G. Friedrich, P. Soriano, Genes Dev 5, 1513 (1991), and mouse ES cells described in US Patent No. 6,190,910. Many other mouse ES lines are available from Jackson Laboratories (Bar Harbor, Maine).
  • human embryonic stem cells include those available through the following suppliers: Arcos Bioscience, Inc., Foster City, California, CyThera, Inc., San Diego, California, BresaGen, Inc., Athens, Georgia, ES Cell International, Melbourne, Australia, Geron Corporation, Menlo Park, California, Goteborg University, Goteborg, Sweden, Karolinska Institute, Sweden, Maria Biotech Co. Ltd.
  • embryonic stem cells are described in the following U.S. patents and published patent applications: 6,245,566; 6,200,806; 6,090,622; 6,331,406; 6,090,622; 5,843,780; 20020045259; 20020068045.
  • the human ES cells are selected from the list of approved cell lines provided by the National Institutes of Health and accessible at http://escr.nih.gov.
  • an embryonic stem cell line is selected from the group consisting of: the WA09 line obtained from Dr. J. Thomson (Univ. of Wisconsin) and the UC01 and UC06 lines, both on the current NLH registry.
  • a stem cell for use in disclosed methods is a stem cell of neural or neuroendocrine origin, such as a stem cell from the central nervous system (see, for example US Patent Nos. 6,468,794; 6,040,180; 5,753,506; 5,766,948), neural crest (see, for example, US Patent Nos. 5,589,376; 5,824, 489), the olfactory bulb or peripheral neural tissues (see, for example, Published US Patent Applications 20030003574; 20020123143; 20020016002 and Gritti et al. 2002 J Neurosci 22(2):437-45), the spinal cord (see, for example, US Patent Nos.
  • a neural stem cell is obtained from a peripheral tissue or an easily healed tissue from a patient in need of cells that produce a pancreatic hormone, thereby providing an autologous population of cells for transplant.
  • hematopoietic or mesenchymal stem cells may be employed in a disclosed method.
  • HSCs marrow-derived hematopoietic
  • MSCs mesenchymal stem cells
  • Purified HSCs not only give rise to all cells in blood, but can also develop into cells normally derived from endoderm, like hepatocytes (Krause et al., 2001, Cell 105: 369-77; Lagasse et al, 2000 Nat Med 6: 1229-34).
  • MSCs appear to be similarly multipotent, producing progeny that can, for example, express neural cell markers (Pittenger et al., 1999 Science 284: 143-7; Zhao et al., 2002 Exp Neurol 174: 11-20).
  • Examples of hematopoietic stem cells include those described in US Patent Nos. 4,714,680; 5,061,620; 5,437,994; 5,914,108; 5,925,567; 5,763,197; 5,750,397; 5,716,827; 5,643,741; 5,061,620.
  • mesenchymal stem cells include those described in US Patent Nos. 5,486,359; 5,827,735; 5,942,225; 5,972,703, those described in PCT publication nos. WO 00/53795; WO 00/02654; WO 98/20907, and those described in Pittenger et al. and Zhao et al., supra.
  • Stem cell lines are preferably derived from mammals, such as rodents (e.g. mouse or rat), primates (e.g. monkeys, chimpanzees or humans), pigs, and ruminants (e.g. cows, sheep and goats), and particularly from humans.
  • stem cells are derived from an autologous source or an HLA-type matched source.
  • stem cells may be obtained from a subject in need of pancreatic hormone-producing cells (e.g. diabetic patients in need of insulin- producing cells) and cultured by a method described herein to generate autologous insulin-producing cells.
  • stem cells are easily obtained from a subject, such as stem cells from muscle tissue, stem cells from skin (dermis or epidermis) and stem cells from fat.
  • Insulin-producing cells may also be derived from banked stem cell sources, such as banked amniotic epithelial stem cells or banked umbilical cord blood cells.
  • banked stem cell sources such as banked amniotic epithelial stem cells or banked umbilical cord blood cells.
  • a s tem c ell m ay b e d erived from a c ell fusion or dedifferentiation process such as described in the following US patent application: 20020090722, and in the following PCT applications: WO200238741, WO0151611, WO9963061, WO9607732.
  • a stem cell line should be compliant with good tissue practice guidelines set for the by the U.S. Food and Drug Administration (FDA) or equivalent regulatory agency in another country. Methods to develop such a cell line may include donor testing, and avoidance of exposure to non-human cells and products during derivation of the stem cell lines.
  • FDA Food and Drug Administration
  • the stem cell line can be prepared and used without the use of a feeder layer or any type of virus or viral vector.
  • both the stem cells and differentiated cells of the methods and compositions disclosed herein have a wild-type genotype, meaning that the genotype of the cells is a genotype that may be found in a subject organism naturally.
  • the genotype of the cells is a genotype that may be found in a subject organism naturally.
  • cells having chromosomal rearragements as a result of culture treatments are not cells having a wild-type genotype.
  • cells that have been transfected with an integrating nucleic acid construct will not (except in cases of perfect excision) have a wild-type genotype.
  • the term "genotype" does not refer to peripheral modifications to the genomic nucleic acids, such as methylation, and therefore, cells having a naturally occurring genetic makeup may have unnatural phenotypes as an effect of changes in methylation or other modifications.
  • Certain embodiments o f the methods d isclosed h erein are advantageous in part because they permit the generation of insulin-producing cell compositions from starting materials, such as certain stem cell lines, that are available, as a practical matter, in sufficient quantities for formation of a therapeutically e ffective insulin- producing implant.
  • starting materials such as certain stem cell lines
  • fetal pancreatic tissue and particularly human fetal pancreatic tissue
  • an insulin-producing cell is exposed to an additional culture condition.
  • insulin-producing cells may be treated with any of the various agents, and functional analogs thereof, that are known to stimulate insulin production or beta-cell proliferation.
  • agents include IGF-1 (e.g. at a concentration of lOng/ml), glucagon-like peptides (e.g. GLP-1), exendin-4, HGF, and reagents that increase cAMP levels, such as membrane permeable forms of cAMP and forskolin.
  • IGF-1 e.g. at a concentration of lOng/ml
  • glucagon-like peptides e.g. GLP-1
  • exendin-4 e.g. HGF
  • HGF reagents that increase cAMP levels, such as membrane permeable forms of cAMP and forskolin.
  • the application provides insulin-producing cell compositions produced according to any of the methods disclosed herein.
  • Insulin- producing cell compositions may be in any form, including, preferably, in insulin- producing cell clusters, but optionally in dispersed cell suspensions, confluent cell cultures, seeded on a matrix or other cell support, etc.
  • the invention relates to insulin-producing cell compositions in which at least about 50% of the cells are positive for insulin production, optionally at least 75% of the cells are positive for insulin production and preferably at least 85%, 90% or 95% of the cells are positive for insulin production.
  • most of the cells, and preferably greater than 80%), 90% or 95% of the cells, that produce insulin are negative for other pancreatic hormones that are not naturally produced by native pancreatic insulin producing cells, such as glucagon.
  • an insulin-producing cell composition comprises at least about 1000 nano grams (ng) of insulin per milligram of total protein, optionally at least about 5000 nano grams of insulin per milligram of total protein and preferably at least about 10000 nanograms of insulin per milligram of total protein.
  • the insulin-producing cell composition comprises islet-like cell clusters of roughly 300-400 ⁇ m in diameter, the clusters produce greater than 0.2 ng of insulin per hour, and preferably greater than 0.5 ng of insulin per hour.
  • insulin production by the insulin-producing cell composition is stimulated by exposure to glucose.
  • insulin-producing cell compositions comprise cells that are positive for one or more of the following markers: insulin (any of the various chains), islet-1, PDX1, GLUT2, glucokinase, a cdk inhibitor, and a tight junction protein, such as a connexin. h certain embodiments, at least about 50% of the cells in an insulin-producing cell composition are not proliferative. Proliferating cells may be detected by a variety of ways known in the art, including staining with Ki67, a nuclear marker of proliferating cells, or incorporation of labeled nucleotide (e.g. tritiated thymidine or bromodeoxyuridine).
  • insulin- producing cell c ompositions do not form neoplastic growths when implanted in a subject. It is understood that biological systems are tremendously variable and, depending on host and implant characteristics, even a very safe insulin-producing cell composition is likely to form, or appear to form, a neoplastic growth at some low frequency.
  • the insulin-producing cell compositions of the invention produce a neoplastic growth in a fewer than 30% of implanted subject, optionally in fewer than 1% of implanted subjects and preferably in fewer than 0.1% of implanted subjects.
  • the application provides methods for ameliorating, in a subject, a condition related to insufficient pancreatic function by administering to the subject an effective amount of an insulin producing cell composition.
  • administration of a BMP signaling activator may be used to ameliorate a condition related to insufficient pancreatic function.
  • administration of a BMP signaling inhibitor may be used to ameliorate a condition related to insufficient pancreatic function. The benefit from an inhibitor versus an activator of BMP signaling may depend on whether regeneration of insulin-producing cells is limited by a failure in maturation of beta cell precursor cells, or a failure in the formation of beta cell precursor cells.
  • a subject may be administered a BMP signaling inhibitor followed by an activator.
  • BMP signaling inhibitors or activators may be coadministered with insulin-producing cells.
  • a sufficient amount of one or more of the above therapeutic compositions is administered to a subject to cause an increase in blood insulin levels or an improvement in glucose homeostasis.
  • Glucose homeostasis may be tested by administering a dose of glucose and monitoring the kinetics with which blood glucose levels decline.
  • Conditions related to insufficient pancreatic function include the various forms of diabetes mellitus (e.g.
  • an insulin-producing cell composition may not produce a permanent ameliorating effect, and periodic dosing, such as on a weekly, monthly or yearly basis may be beneficial.
  • an effective dose of insulin-producing cell composition comprises administering at least about one islet-like cell cluster of the invention (or an equivalent number of cells) per islet that is naturally present in the subject organism.
  • mice have about 300-500 islets
  • rats have about 3000-5000 islets
  • humans have about 1,000,000 islets
  • a preferred dosage is about 300-500 islet-like cell clusters for a mouse, about 3000- 5000 islet-like cell clusters for a rat and about 1,000,000 islet-like cell clusters for a human.
  • the number of islets per organism is proportional to average body mass (20-30 grams, mouse, 200-300 grams, rat, 60-70 kilograms, human) and it may be desirable to administer a dosage that is proportional to body mass of the subject.
  • a dosage may be increased proportionally.
  • the invention relates to therapeutic compositions comprising insulin-producing cell compositions and methods for making such therapeutic compositions.
  • Therapeutic compositions include an insulin-producing cell compostions disclosed herein and/or made by the methods disclosed herein, as well as mixtures comprising such insulin-producing cell compositions and a therapeutic excipient.
  • therapeutic excipients include matrices, scaffolds or other substrates to which cells may attach (optionally formed as solid or hollow beads, tubes, or membranes), as well as reagents that are useful in facilitating administration (e.g. buffers and salts), preserving the cells (e.g. chelators such as sorbates, EDTA, EGTA, or quaternary amines or other antibiotics), or promoting engraftment.
  • Cells may be encapsulated in a membrane to avoid immune rejection. By manipulation of the membrane permeability, so as to allow free diffusion of glucose and insulin back and forth through the membrane, yet block passage of antibodies and lymphocytes, normoglycemia may be maintained (Sullivan et al. (1991) Science 252:718).
  • hollow fibers containing cells may be immobilized in a polysaccharide alginate. (Lacey et al. (1991) Science 254:1782). Cells may be placed in microcapsules composed of alginate or polyacrylates. (Lim et al. (1980) Science 210:908; O'Shea et al. (1984) Biochim. Biochys. Acta.
  • Patent No. 4,391,909 U.S. Patent No. 4,353,888; Sugamori et al. (1989) Trans. Am.
  • the site of implantation of insulin-producing cell compositions may be selected by one of skill in the art. In general, such as site preferably has adequate blood perfusion to allow the cells to sense blood conditions and secrete hormones and other factors into the general circulation. Exemplary implantation sites include the liver (via portal vein injection), the peritoneal cavity, the kidney capsule and the pancreas.
  • the application provides methods for assessing the effectiveness of a test agent for modulating the development of insulin producing cells. Such methods may be used to screen libraries of test agents. In general, a method of this type involves assessing the ability of the test agent to interfere with or promote BMP pathway signaling. Two non-limiting categories of assays include biochemical assays and cell-based assays.
  • a test agent may be mixed with a BMP pathway polypeptide; and a test agent that binds the BMP pathway polypeptide specifically and with an affinity of at least 10 "4 M, and preferably 10 "5 , 10 "6 , 10 "7 or less, has an increased 1 ikelihood o f b eing e ffective for m odulating t he d evelopment o f i nsulin producing cells.
  • An agent that binds may inhibit signaling, as in the case of an agent that b inds and t itrates away a BMP family m ember.
  • an a gent that binds may activate, as in the case of an agent that binds to an ActRIIA or ActRIiB receptor and mimics activation by a BMP family member.
  • Test agents may be assessed in an assay system that measures binding between a BMP family member, such as a GDF8/GDF11 family member, and a receptor, such as an ActRIIA or ActRIIB.
  • a cell is cultured with the test agent; and an activity of a BMP signaling pathway is detected, wherein a test agent that increases the activity of a BMP signaling pathway has an increased likelihood of being effective for increasing insulin production in a cell.
  • Test agents for promoting formation of beta cell precursor cells may similarly be tested by determining whether they cause a decreases in the activity of a BMP signaling pathway.
  • Cells for use in such assays preferably express an ActRIIA and/or ActRIIB receptor.
  • assay cells comprise a reporter gene, wherein expression of the reporter gene is positively or negatively regulated by a BMP signaling pathway.
  • a reporter gene may be designed to be activated by Smad4, which is positively regulated by BMP signaling.
  • Smad4 regulated genes include cdk inhibitors, such as pi 5, pi 7, pi 8, p21, p27 and p57.
  • the activity of a BMP signaling pathway may also be assessed by measuring a change in one or more components of the BMP signaling pathway. For example, phosphorylation of one or more pathway proteins, such as ActRIIA, ActRIIB, Smad2 and Smad3, may be evaluated. 7. Methods for Assessing Candidate Islet Cell Differentiation Factors and Other Test Compounds
  • the application provides methods for obtaining beta cell precursor cell populations as well as insulin-producing cells, and such cells may be used for a variety of purposes, such as the identification of markers for these cell types.
  • the application provides methods for assessing whether a test agent has beta cell precursor cell differentiation activity.
  • An exemplary embodiment of such a method may comprise contacting beta cell precursor cells with a test agent and detecting a beta cell marker.
  • a test agent that stimulates the formation of cells expressing islet cell markers has beta cell precursor cell differentiation activity activity.
  • beta cell marker is intended to include any phenotype that is distinctive of one or more islet cell types, including various protein, nucleic acid, morphological, biochemical (e.g. metabolic or transport) or other phenotypes.
  • beta cell markers include the following polypeptides or the corresponding RNA transcript: insulin (any of the various chains, including, for example, C-peptide, mature insulin or proinsulin), GLUT2, glucokinase, PDX-1, LAPP, SUR1, PCI/3, PC2, KLR6.2, pancreatic polypeptide, somatostatin, glucagon, glucokinase and C-peptide.
  • insulin any of the various chains, including, for example, C-peptide, mature insulin or proinsulin
  • GLUT2 glucokinase
  • PDX-1 PDX-1
  • LAPP LAPP
  • SUR1 PCI/3
  • PC2, KLR6.2 pancreatic polypeptide
  • somatostatin glucagon
  • glucokinase and C-peptide C-peptide.
  • the subject cells can be used to screen various compounds or natural products, such as small molecules or growth factors.
  • the efficacy of the test agent can be assessed by generating dose response
  • methods of the application relate to the identification of pancreatic developmental markers.
  • expression patterns of established markers may be monitored at one or more stages of differentiation of stem cells into beta cell precursors and insulin-producing cells. Markers may be assessed using standard methods, including Northern blotting, RT- PCR, in situ hybridization (ISH), immunohistochemistry (IHC) as well as nucleic acid or protein array or microarray-based methods.
  • monitoring production of one or more gene products will be useful to identify candidate cell-surface proteins for FACS-based purification strategies for insulin- producing cell precursors.
  • the application provides methods for identifying affinity reagent that bind to cells at various stages of pancreatic development.
  • Affinity reagents include antibodies, and preferably monoclonal antibodies, targeting peptides (e.g. peptides selected from a high diversity phage display library), RNA or DNA aptamers.
  • the term "antibody” as used herein is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments thereof which are also specifically reactive with a vertebrate, e.g., mammalian, protein.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility and/or interaction with a specific epitope of interest.
  • the term includes segments of proteolytically-cleaved or recombinan ly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein.
  • proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab' , Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker.
  • the scFv's may be covalently or non-covalently linked to form antibodies having two or more binding sites.
  • antibody includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies.
  • beta cell precursors or insulin producing cells may be used to screen a plurality of affinity reagents.
  • the cells themselves may be used for the screening, or membrane or protein extracts may be used.
  • cell surface proteins may be selectively labeled and used to screen a plurality of affinity reagents.
  • the plurality of affinity reagents to be screened is a library of monoclonal antibodies.
  • An affinity reagent detected as binding to a cell such as an beta cell precursor cell may be tested on tissue samples for capability to detect particular subpopulations of pancreatic or pre-pancreatic cells, and it is of particular interest t o i dentify affinity r eagents t hat are u seful i n t he i dentification o f n atural populations of cells that are precursors of beta cells or other islet cells.
  • Yet another aspect of the present application provides methods for screening various compounds for their ability to modulate insulin-producing cells, such as, for example, by affecting growth, proliferation, maturation or differentiation, or by affecting i nsulin p roduction, s ecretion o r s torage, a s w ell a s c ompounds that m ay improve graft performance (e.g. result in a longer-lasting graft, improved insulin production, or changes in proteins that interact with the host immune system).
  • the subject cells can be used to screen various compounds or natural products, such as small molecules or growth factors.
  • insulin-producing cells may be used to test the activity of compounds/factors to promote survival and maturation, and further, since certain cells produced according to methods disclosed herein have one or more properties of islet cells, specifically ⁇ -cells, such cells may be used to identify factors (or genes) that regulate production, processing, storage, secretion, and degradation of insulin or other relevant proteins (like LAPP, glucagon, including pro-glucagon, GLPs, etc) produced in pancreatic islets.
  • an insulin-producing cell may be modified, such as by genetic modification, to become hyperproliferative.
  • hyperproliferative cells may be contacted with compounds to identify, for example, anti-proliferative and anti-neoplastic agents (e.g. agents that inhibit cell growth or promote cell death).
  • anti-proliferative and anti-neoplastic agents e.g. agents that inhibit cell growth or promote cell death.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the compound.
  • a control assay can also be performed to provide a baseline for comparison.
  • Identification of the progenitor cell population(s) amplified in response to a given test agent can be carried out according to such phenotyping as described above. Assays such as those described above may be carried out in vitro (e.g. with cells in culture) or in vivo (e.g. with cell implanted in a subject).
  • the application relates to methods for identifying a cell that has the potential to develop into a pancreatic cell, and particularly an insulin-producing cell.
  • the method comprises providing a stem cell line, or other multipotent cell line, and differentiating the cell line so as to obtain an insulin-producing cell composition.
  • the differentiating cells are mixed with a cell of interest.
  • the differentiation of the cell of interest may then be assessed.
  • a cell of interest that is able to differentiate into an insulin-producing cell is a cell that has the potential to develop into an insulin-producing cell.
  • the cell may be assessed for the production of other pancreatic products, such as glucagons, to identify cells that have the potential to develop into other types of pancreatic cells.
  • a pancreatic tissue e.g. ductal tissue, adult pancreatic tissue, fetal pancreatic tissue, etc.
  • clumps of cells or single cells are used as the cell of interest in the above method embodiments, thereby permitting a rapid screen of pancreatic cells for candidate pancreatic progenitors.
  • insulin-producing cell compositions and methods for generating such compositions may be used to assess the developmental potential of a cell of interest.
  • the developmental potential of a cell of interest may be determined by mixing the cell of interest with cells during the process of making beta precursor or insulin-producing cells (i.e. co-culturing). The cell of interest is then tracked (for example by a transgenic marker) to determine the types of cells that arise from it.
  • the cell of interest is mixed with differentiating neural or neuroendocrine stem cells.
  • c ulture sy stems for m aking i nsulin-producing c ell compositions may be used as part of an assay to identify candidate pancreatic endocrine p recursor c ells.
  • C urrent e vidence s uggests t hat s uch p recursors e xist a s single cells or small cell clusters within or closely associated with pancreatic epithelium.
  • cell compositions in the process of differentiating i nto b eta c ell p recursor c ells o r i nsulin-producing c ells p rovide t he appropriate cellular microenvironment to permit pancreas-derived endoderm to integrate and differentiate.
  • cells of a pancreatic tissue are fractionated and mixed, either as populations of c ells or as single c ells, into cells being differentiated into insulin-producing cell compositions.
  • Cells of the pancreatic tissue that develop into insulin-producing cells are candidate pancreatic stem cells.
  • a fraction of cells that are in the process of differentiating into insulin-producing cell compositions may be used in the culture medium of the cells of interest. Fractions that may be used include conditioned media or other preparations of secreted material, extracellular matrix, membrane preparations, total soluble protein, soluble cellular protein and other portions of cells that are in the process of differentiating into beta cell precursors or insulin-producing cells.
  • GDF-11 Growth and differentiation factor 11
  • GDF-11 is a recently identified member of the TGF- ⁇ ligand superfamily (Nakashima et al., 1999; Gamer et al, 1999). Homozygous null GDF-11 mutants manifest several defects including skeletal transformations, and abnormal Hox gene expression that resemble defects observed in ActRIIB mutants (McPherron and Lee, 1999). GDF-11 expression in pancreatic epithelium begins by E9.5-10 in pancreatic epithelium, continues there throughout gestation, and is later abundantly expressed in isolated adult islets.
  • hyposplenism with splenic malformations (2) defects in axial patterning of stomach epithelium and mesenchyme, and (3) severe pancreatic defects including islet hypoplasia, defects in islet cell differentiation, increased accumulation of neurogenin3 -expressing cells (presumptive islet cell precursors) during embryonic pancreas formation, and reduced ⁇ -cell mass.
  • morphogenetic defects in p ancreas development are s trikingly s imilar i n GDF-11 and ActRIIB mutants (Kim et al., 2000).
  • GDF-11 as a candidate TGF- ⁇ ligand that regulates development of pancreatic islet precursor cells and subsequent maturation of pancreatic ⁇ -cells to functioning insulin- producing and secreting cells. Data are shown in Figures 1-4 and 6.
  • Smad2 activity is regulated both by ActRIIA and ActRIIB.
  • Smad2 encodes a transcription factor which is phosphorylated by type I activin or TGF- ⁇ receptors after ligand binding to type II and type I receptors, hi mice, Smad2, ActRIIA, ActRIIB and other TGF- ⁇ signaling components are expressed in embryonic pancreas, and later in adult islets.
  • Smad2 was investigated roles of Smad2 in pancreas development and function.
  • mice heterozygous for Smad2 mutation are viable, and similar to ActRILB-/- and ActRIIA+/- IIB+/- mutant mice, have (1) increased production of cells in the embryonic pancreas expressing neurogenin3, a marker of pancreatic islet precursors (2) increased numbers of immature pancreatic ⁇ -cells late in gestation (e.g., cells expressing the transcription factor Nkx6.1 but not insulin) (3) evidence of impaired islet maturation after birth, with islet hypoplasia and reduced ⁇ -cell mass, (4) normal islet architecture, without evidence of other organ malformations (5) impaired glucose tolerance, and (6) inadequate blood insulin levels. These results are encompassed in a manuscript in preparation (Harmon et al).
  • Example 3 In vitro studies of noggin, chordin, GDF11 and GDF8 activity on mouse ES cells during formation of insulin-producing cell clusters (TPCCs .
  • GDF8 (which is -80% identical to GDF11 in the mature region and has similar in vitro activities in a neural cell development assay as shown by published studies from the laboratory of Thomas Jessell, Columbia University) promotes insulin p roduction in mouse ES c ell-derived LPCCs. In duplicate experiments, we detect approximately 6000 ng insulin/mg protein produced by mouse embryoid bodies treated with a sequence of growth factors including GDF8. Data are shown in Figure 7.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne, entre autres, des méthodes permettant d'augmenter la production d'insuline dans des cellules par mise en contact de ces cellules avec un élément de la famille BMP et/ou un activateur d'une voie BMP. Cette invention concerne également des méthodes permettant d'augmenter la production de précurseurs de cellules Bêta par mise en contact de cellules adéquates avec un inhibiteur d'une voie BMP.
PCT/US2004/004639 2003-02-14 2004-02-17 Methodes et compositions permettant de moduler le developpement de cellules souches Ceased WO2004073633A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/545,582 US20070155661A1 (en) 2003-02-14 2004-02-17 Methods and compositions for modulating the development of stem cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US44767303P 2003-02-14 2003-02-14
US60/447,673 2003-02-14

Publications (2)

Publication Number Publication Date
WO2004073633A2 true WO2004073633A2 (fr) 2004-09-02
WO2004073633A3 WO2004073633A3 (fr) 2005-12-15

Family

ID=32908479

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/004639 Ceased WO2004073633A2 (fr) 2003-02-14 2004-02-17 Methodes et compositions permettant de moduler le developpement de cellules souches

Country Status (2)

Country Link
US (1) US20070155661A1 (fr)
WO (1) WO2004073633A2 (fr)

Cited By (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007099345A1 (fr) * 2006-03-02 2007-09-07 Betagenon Ab Utilisation médicale de bmp-2 et/ou bmp-4
WO2010051213A1 (fr) * 2008-10-31 2010-05-06 Centocor Ortho Biotech Inc. Différenciation de cellules souches embryonnaires humaines en la lignée endocrine pancréatique
EP2297319A4 (fr) * 2008-06-03 2011-09-07 Viacyte Inc Facteurs de croissance pour la production de l'endoderme définitif
US20120190112A1 (en) * 2008-06-30 2012-07-26 Janet Davis Differentiation of Pluripotent Stem Cells
AU2011203583B2 (en) * 2004-07-09 2013-03-07 Viacyte, Inc. Methods for identifying factors for differentiating definitive endoderm
US8647873B2 (en) 2004-04-27 2014-02-11 Viacyte, Inc. PDX1 expressing endoderm
EP2828289A4 (fr) * 2012-03-19 2016-01-06 Brigham & Womens Hospital Facteur de différenciation de croissance (gdf) destiné au traitement d'insuffisance cardiaque diastolique
US9725699B2 (en) 2006-04-28 2017-08-08 Lifescan, Inc. Differentiation of human embryonic stem cells
US9732318B2 (en) 2003-12-23 2017-08-15 Viacyte, Inc. Preprimitive streak and mesendoderm cells
US9744195B2 (en) 2007-07-31 2017-08-29 Lifescan, Inc. Differentiation of human embryonic stem cells
US9752126B2 (en) 2008-10-31 2017-09-05 Janssen Biotech, Inc. Differentiation of human pluripotent stem cells
US9752125B2 (en) 2010-05-12 2017-09-05 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9845460B2 (en) 2008-04-24 2017-12-19 Janssen Biotech, Inc. Treatment of pluripotent cells
US9951314B2 (en) 2010-08-31 2018-04-24 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9969972B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Pluripotent stem cell culture on micro-carriers
US9969981B2 (en) 2010-03-01 2018-05-15 Janssen Biotech, Inc. Methods for purifying cells derived from pluripotent stem cells
US9969973B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Methods and compositions for cell attachment and cultivation on planar substrates
US9969982B2 (en) 2007-11-27 2018-05-15 Lifescan, Inc. Differentiation of human embryonic stem cells
US10006006B2 (en) 2014-05-16 2018-06-26 Janssen Biotech, Inc. Use of small molecules to enhance MAFA expression in pancreatic endocrine cells
US10017566B2 (en) 2013-11-12 2018-07-10 The Brigham And Women's Hospital, Inc. Growth differentiation factor (GDF) for treatment of diastolic heart failure
US10066203B2 (en) 2008-02-21 2018-09-04 Janssen Biotech Inc. Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
US10066210B2 (en) 2012-06-08 2018-09-04 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells
US10076544B2 (en) 2009-07-20 2018-09-18 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US10138465B2 (en) 2012-12-31 2018-11-27 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells using HB9 regulators
US10259861B2 (en) 2007-02-02 2019-04-16 Acceleron Pharma Inc. Variants derived from ActRIIB and uses therefor
US10316293B2 (en) 2007-07-01 2019-06-11 Janssen Biotech, Inc. Methods for producing single pluripotent stem cells and differentiation thereof
US10344264B2 (en) 2012-12-31 2019-07-09 Janssen Biotech, Inc. Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells
US10358628B2 (en) 2011-12-22 2019-07-23 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US10370644B2 (en) 2012-12-31 2019-08-06 Janssen Biotech, Inc. Method for making human pluripotent suspension cultures and cells derived therefrom
US10377989B2 (en) 2012-12-31 2019-08-13 Janssen Biotech, Inc. Methods for suspension cultures of human pluripotent stem cells
US10420803B2 (en) 2016-04-14 2019-09-24 Janssen Biotech, Inc. Differentiation of pluripotent stem cells to intestinal midgut endoderm cells
US10421942B2 (en) 2003-12-23 2019-09-24 Viacyte, Inc. Definitive endoderm
EP3399998A4 (fr) * 2016-01-06 2019-11-06 President and Fellows of Harvard College Un traitement par le gdf11 prévient la prise de poids, améliore la tolérance au glucose, et diminue la stéatose hépatique
US10704025B2 (en) 2009-12-23 2020-07-07 Janssen Biotech, Inc. Use of noggin, an ALK5 inhibitor and a protein kinase c activator to produce endocrine cells
US10968282B2 (en) 2009-06-08 2021-04-06 Acceleron Pharma Inc. Methods for screening compounds for increasing thermogenic adipocytes
US11155791B2 (en) 2008-08-14 2021-10-26 Acceleron Pharma Inc. Methods for treating anemia in a subject in need thereof
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11612639B2 (en) 2013-04-08 2023-03-28 President And Fellows Of Harvard College Methods and compositions for rejuvenating skeletal muscle stem cells
US11613727B2 (en) 2010-10-08 2023-03-28 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US11634677B2 (en) 2016-06-07 2023-04-25 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US11667876B2 (en) 2013-11-16 2023-06-06 Terumo Bct, Inc. Expanding cells in a bioreactor
US11667881B2 (en) 2014-09-26 2023-06-06 Terumo Bct, Inc. Scheduled feed
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US12043823B2 (en) 2021-03-23 2024-07-23 Terumo Bct, Inc. Cell capture and expansion
US12152699B2 (en) 2022-02-28 2024-11-26 Terumo Bct, Inc. Multiple-tube pinch valve assembly
US12234441B2 (en) 2017-03-31 2025-02-25 Terumo Bct, Inc. Cell expansion
USD1099116S1 (en) 2022-09-01 2025-10-21 Terumo Bct, Inc. Display screen or portion thereof with a graphical user interface for displaying cell culture process steps and measurements of an associated bioreactor device

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8017395B2 (en) 2004-12-17 2011-09-13 Lifescan, Inc. Seeding cells on porous supports
AU2006255183B2 (en) 2005-06-08 2012-02-02 Centocor, Inc. A cellular therapy for ocular degeneration
US8106004B2 (en) * 2006-07-28 2012-01-31 Children's Memorial Hospital Methods of inhibiting tumor cell aggressiveness using the microenvironment of human embryonic stem cells
US7666423B2 (en) * 2006-07-28 2010-02-23 Children's Memorial Hospital Methods of inhibiting tumor cell aggressiveness using the microenvironment of human embryonic stem cells
US20090298178A1 (en) * 2008-06-03 2009-12-03 D Amour Kevin Allen Growth factors for production of definitive endoderm
EP2456859A4 (fr) 2009-07-20 2015-03-18 Janssen Biotech Inc Différentiation de cellules souches embryonnaires humaines
PL2456858T3 (pl) 2009-07-20 2019-01-31 Janssen Biotech, Inc Różnicowanie ludzkich embrionalnych komórek macierzystych
EP2516626B1 (fr) 2009-12-23 2017-05-10 Janssen Biotech, Inc. Différenciation de cellules souches embryonnaires humaines
MX355077B (es) 2010-08-31 2018-04-03 Janssen Biotech Inc Diferenciacion de celulas madre embrionarias humanas.
MX348537B (es) 2010-08-31 2017-06-07 Janssen Biotech Inc Diferencia de celulas madre pluripotentes.
SG11201405052RA (en) 2012-03-07 2014-10-30 Janssen Biotech Inc Defined media for expansion and maintenance of pluripotent stem cells
WO2016179106A1 (fr) * 2015-05-01 2016-11-10 University Of Miami Procédés et compositions de conversion de tissu pancréatique non endocrine en cellules produisant de l'insuline
CA2983845C (fr) * 2017-10-26 2024-01-30 University Of Copenhagen Generation de cellules beta reagissant au glucose

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030167492A1 (en) * 1994-07-08 2003-09-04 Johns Hopkins University School Of Medicine Transgenic non-human animals expressing a gdf-11 dominant negative polypeptide, and methods of making and using same
GB2385052A (en) * 2002-02-05 2003-08-13 Leuven K U Res & Dev Treatment of spondyloarthropathies

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HORI Y. ET AL: 'Differentiation of insulin-producing cells from human neural progenitor cells.' PLOS MED. vol. 2, no. 4, April 2005, pages 347 - 356 *
HORI Y. ET AL: 'Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells.' PROC NATL ACAD SCI. vol. 99, no. 25, December 2002, pages 16105 - 16110 *
KU H.T. ET AL: 'Committing embryonic stem cells to early endocrine pancreas in vitro.' STEM CELLS. vol. 22, no. 7, 2004, pages 1205 - 1217 *
TANG D.Q. ET AL: 'In vivo and in vitro characterization of insulin-producing cells obteined from murine bone marrow.' DIABETES. vol. 53, no. 7, July 2004, pages 1721 - 1732 *

Cited By (88)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9732318B2 (en) 2003-12-23 2017-08-15 Viacyte, Inc. Preprimitive streak and mesendoderm cells
US10179902B2 (en) 2003-12-23 2019-01-15 Viacyte, Inc. Methods of making human primitive ectoderm cells
US10421942B2 (en) 2003-12-23 2019-09-24 Viacyte, Inc. Definitive endoderm
US11667889B2 (en) 2003-12-23 2023-06-06 Viacyte, Inc. Methods of making human primitive ectoderm cells
US10550367B2 (en) 2003-12-23 2020-02-04 Viacyte, Inc. Methods of making human primitive ectoderm cells
US8647873B2 (en) 2004-04-27 2014-02-11 Viacyte, Inc. PDX1 expressing endoderm
US9222069B2 (en) 2004-04-27 2015-12-29 Viacyte, Inc. Methods for making anterior foregut endoderm
US11746323B2 (en) 2004-04-27 2023-09-05 Viacyte, Inc. PDX1 positive foregut endoderm cells and methods of production
US10465162B2 (en) 2004-04-27 2019-11-05 Viacyte, Inc. Anterior endoderm cells and methods of production
AU2011203583B2 (en) * 2004-07-09 2013-03-07 Viacyte, Inc. Methods for identifying factors for differentiating definitive endoderm
WO2007099345A1 (fr) * 2006-03-02 2007-09-07 Betagenon Ab Utilisation médicale de bmp-2 et/ou bmp-4
US9725699B2 (en) 2006-04-28 2017-08-08 Lifescan, Inc. Differentiation of human embryonic stem cells
EA033623B1 (ru) * 2007-02-02 2019-11-11 Acceleron Pharma Inc ВАРИАНТЫ, ПРОИСХОДЯЩИЕ ИЗ ActRIIB, И ИХ ПРИМЕНЕНИЕ
US10259861B2 (en) 2007-02-02 2019-04-16 Acceleron Pharma Inc. Variants derived from ActRIIB and uses therefor
US10316293B2 (en) 2007-07-01 2019-06-11 Janssen Biotech, Inc. Methods for producing single pluripotent stem cells and differentiation thereof
US9744195B2 (en) 2007-07-31 2017-08-29 Lifescan, Inc. Differentiation of human embryonic stem cells
US10456424B2 (en) 2007-07-31 2019-10-29 Janssen Biotech, Inc. Pancreatic endocrine cells and methods thereof
US9969982B2 (en) 2007-11-27 2018-05-15 Lifescan, Inc. Differentiation of human embryonic stem cells
US11001802B2 (en) 2008-02-21 2021-05-11 Nunc A/S Surface of a vessel with polystyrene, nitrogen, oxygen and a static sessile contact angle for attachment and cultivation of cells
US10066203B2 (en) 2008-02-21 2018-09-04 Janssen Biotech Inc. Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
US9845460B2 (en) 2008-04-24 2017-12-19 Janssen Biotech, Inc. Treatment of pluripotent cells
EP2993226A1 (fr) * 2008-06-03 2016-03-09 Viacyte, Inc. Facteurs de croissance pour la production d'un endoderme définitif
EP2297319A4 (fr) * 2008-06-03 2011-09-07 Viacyte Inc Facteurs de croissance pour la production de l'endoderme définitif
US10233421B2 (en) 2008-06-30 2019-03-19 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
US10351820B2 (en) 2008-06-30 2019-07-16 Janssen Biotech, Inc. Methods for making definitive endoderm using at least GDF-8
RU2673750C2 (ru) * 2008-06-30 2018-11-29 Сентокор Орто Байотек Инк. Дифференцирование плюрипотентных стволовых клеток
US20120190112A1 (en) * 2008-06-30 2012-07-26 Janet Davis Differentiation of Pluripotent Stem Cells
US11168311B2 (en) 2008-08-14 2021-11-09 Acceleron Pharma Inc. Methods for treating anemia in a subject in need thereof
US11162085B2 (en) 2008-08-14 2021-11-02 Acceleron Pharma Inc. Methods for treating anemia in a subject in need thereof
US11155791B2 (en) 2008-08-14 2021-10-26 Acceleron Pharma Inc. Methods for treating anemia in a subject in need thereof
WO2010051213A1 (fr) * 2008-10-31 2010-05-06 Centocor Ortho Biotech Inc. Différenciation de cellules souches embryonnaires humaines en la lignée endocrine pancréatique
US9752126B2 (en) 2008-10-31 2017-09-05 Janssen Biotech, Inc. Differentiation of human pluripotent stem cells
CN102333862B (zh) * 2008-10-31 2018-04-27 詹森生物科技公司 人胚胎干细胞向胰腺内分泌谱系的分化
EP3517605A1 (fr) * 2008-10-31 2019-07-31 Janssen Biotech, Inc. Différenciation de cellules souches embryonnaires humaines
US9969972B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Pluripotent stem cell culture on micro-carriers
US9969973B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Methods and compositions for cell attachment and cultivation on planar substrates
US10968282B2 (en) 2009-06-08 2021-04-06 Acceleron Pharma Inc. Methods for screening compounds for increasing thermogenic adipocytes
US10076544B2 (en) 2009-07-20 2018-09-18 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US10471104B2 (en) 2009-07-20 2019-11-12 Janssen Biotech, Inc. Lowering blood glucose
US10704025B2 (en) 2009-12-23 2020-07-07 Janssen Biotech, Inc. Use of noggin, an ALK5 inhibitor and a protein kinase c activator to produce endocrine cells
US10329534B2 (en) 2010-03-01 2019-06-25 Janssen Biotech, Inc. Methods for purifying cells derived from pluripotent stem cells
US9969981B2 (en) 2010-03-01 2018-05-15 Janssen Biotech, Inc. Methods for purifying cells derived from pluripotent stem cells
US9752125B2 (en) 2010-05-12 2017-09-05 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9951314B2 (en) 2010-08-31 2018-04-24 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US11773363B2 (en) 2010-10-08 2023-10-03 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11613727B2 (en) 2010-10-08 2023-03-28 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11746319B2 (en) 2010-10-08 2023-09-05 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US12215354B2 (en) 2011-12-22 2025-02-04 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US11377640B2 (en) 2011-12-22 2022-07-05 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US10358628B2 (en) 2011-12-22 2019-07-23 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US9434779B2 (en) 2012-03-19 2016-09-06 The Brigham And Women's Hospital, Inc. Growth differentiation factor 11 (GDF-11) for treatment of diastolic heart failure
EP2828289A4 (fr) * 2012-03-19 2016-01-06 Brigham & Womens Hospital Facteur de différenciation de croissance (gdf) destiné au traitement d'insuffisance cardiaque diastolique
US10208288B2 (en) 2012-06-08 2019-02-19 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells
US10066210B2 (en) 2012-06-08 2018-09-04 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells
US10947511B2 (en) 2012-12-31 2021-03-16 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells using thyroid hormone and/or alk5, an inhibitor of tgf-beta type 1 receptor
US10370644B2 (en) 2012-12-31 2019-08-06 Janssen Biotech, Inc. Method for making human pluripotent suspension cultures and cells derived therefrom
US10138465B2 (en) 2012-12-31 2018-11-27 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells using HB9 regulators
US10377989B2 (en) 2012-12-31 2019-08-13 Janssen Biotech, Inc. Methods for suspension cultures of human pluripotent stem cells
US10344264B2 (en) 2012-12-31 2019-07-09 Janssen Biotech, Inc. Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells
US11612639B2 (en) 2013-04-08 2023-03-28 President And Fellows Of Harvard College Methods and compositions for rejuvenating skeletal muscle stem cells
US10017566B2 (en) 2013-11-12 2018-07-10 The Brigham And Women's Hospital, Inc. Growth differentiation factor (GDF) for treatment of diastolic heart failure
US11708554B2 (en) 2013-11-16 2023-07-25 Terumo Bct, Inc. Expanding cells in a bioreactor
US11667876B2 (en) 2013-11-16 2023-06-06 Terumo Bct, Inc. Expanding cells in a bioreactor
US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US10006006B2 (en) 2014-05-16 2018-06-26 Janssen Biotech, Inc. Use of small molecules to enhance MAFA expression in pancreatic endocrine cells
US10870832B2 (en) 2014-05-16 2020-12-22 Janssen Biotech, Inc. Use of small molecules to enhance MAFA expression in pancreatic endocrine cells
US12065637B2 (en) 2014-09-26 2024-08-20 Terumo Bct, Inc. Scheduled feed
US11667881B2 (en) 2014-09-26 2023-06-06 Terumo Bct, Inc. Scheduled feed
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
EP3399998A4 (fr) * 2016-01-06 2019-11-06 President and Fellows of Harvard College Un traitement par le gdf11 prévient la prise de poids, améliore la tolérance au glucose, et diminue la stéatose hépatique
US11890321B2 (en) 2016-01-06 2024-02-06 President And Fellows Of Harvard College Method of treating or inhibiting glucose intolerance
EP3949979A1 (fr) * 2016-01-06 2022-02-09 President and Fellows of Harvard College Un traitement par le gdf11 prévient la prise de poids, améliore la tolérance au glucose, et diminue la stéatose hépatique
US10980857B2 (en) 2016-01-06 2021-04-20 President And Fellows Of Harvard College Treatment with GDF11 prevents weight gain, improves glucose tolerance and reduces hepatosteatosis
US10420803B2 (en) 2016-04-14 2019-09-24 Janssen Biotech, Inc. Differentiation of pluripotent stem cells to intestinal midgut endoderm cells
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11999929B2 (en) 2016-06-07 2024-06-04 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11634677B2 (en) 2016-06-07 2023-04-25 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US12077739B2 (en) 2016-06-07 2024-09-03 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US11702634B2 (en) 2017-03-31 2023-07-18 Terumo Bct, Inc. Expanding cells in a bioreactor
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
US12234441B2 (en) 2017-03-31 2025-02-25 Terumo Bct, Inc. Cell expansion
US12359170B2 (en) 2017-03-31 2025-07-15 Terumo Bct, Inc. Expanding cells in a bioreactor
US12043823B2 (en) 2021-03-23 2024-07-23 Terumo Bct, Inc. Cell capture and expansion
US12152699B2 (en) 2022-02-28 2024-11-26 Terumo Bct, Inc. Multiple-tube pinch valve assembly
US12209689B2 (en) 2022-02-28 2025-01-28 Terumo Kabushiki Kaisha Multiple-tube pinch valve assembly
USD1099116S1 (en) 2022-09-01 2025-10-21 Terumo Bct, Inc. Display screen or portion thereof with a graphical user interface for displaying cell culture process steps and measurements of an associated bioreactor device

Also Published As

Publication number Publication date
WO2004073633A3 (fr) 2005-12-15
US20070155661A1 (en) 2007-07-05

Similar Documents

Publication Publication Date Title
US20070155661A1 (en) Methods and compositions for modulating the development of stem cells
Siehler et al. Engineering islets from stem cells for advanced therapies of diabetes
US20070154981A1 (en) Insulin-producing cells derived from stem cells
DE112014002780B4 (de) SC-ß-Zellen und Zusammensetzungen und Verfahren zur Erzeugung der Zellen
US6753153B2 (en) Markers for identification and isolation of pancreatic islet α and β progenitors
CN104411815B (zh) 用于从hPSC生成胰腺祖细胞和功能性β细胞的方法和组合物
US20040121460A1 (en) Differentiation of stem cells to pancreatic endocrine cells
JP2019516384A (ja) オルガノイド作製および疾患モデル化のための組成物および方法
US20040259244A1 (en) Methods, compositions, and growth and differentiation factors for insulin-producing cells
AU2002350352A1 (en) Cellular compositions and methods of making and using them
Parekh et al. Differentiation of human umbilical cord blood-derived mononuclear cells to endocrine pancreatic lineage
AU2003240810A1 (en) Methods, compositions, and growth and differentiation factors for insulin-producing cells
CZ2004696A3 (cs) Endokrinní pankreatická diferenciace buněk stromatu odvozených z adipózní tkáně a její použití
Davies et al. Stemistry: the control of stem cells in situ using chemistry
JP6685327B2 (ja) 膵島移植のための改善された方法
US20240368557A1 (en) Stem cell-derived alpha cells and methods of generating same
US20060093586A1 (en) Generation and differentiation of adult stem cell lines
Bose et al. Regenerative medicine for diabetes: differentiation of human pluripotent stem cells into functional β-cells in vitro and their proposed journey to clinical translation
CN119968460A (zh) 用于在不存在细胞外基质的条件下培养类器官的药物
Sordi et al. Diabetes mellitus: an opportunity for therapy with stem cells?
JP2006075022A (ja) 膵臓ホルモン産生細胞取得方法
WO2004010933A2 (fr) Compositions cellulaires produisant de l'insuline et procedes associes
Racanicchi et al. Neonatal Pig Liver–Derived Progenitors for Insulin-Producing Cells: An In Vitro Study
Sultana et al. Insight into (?) Cell Development to Rebuild Bonafide?-Cells: Reprogramming Verses Differentiation of Stem Cells
Samuelson Sca-1 positive pancreatic progenitor cells: a replacement for transplanted islets

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 10545582

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 10545582

Country of ref document: US